Over the last decade, our ability to cultivate the intestinal protozoan, Cryptosporidium parvum, in cell culture has increased by >500x. However, there still exist multiple fundamental problems associated with cultivating this parasite in vitro. One of these problems is the diversity of medium formulations currently in use, most of which are sub-optimal for growing the parasites in vitro. Formulations which result in the most extensive parasite development complete with both asexual and sexual stages rely on the investigatoradding5-6 key supplements and many laboratories fail to make the modifications resulting in sub-optimal data. The second of these problems, and more importantly, is our inability to produce high numbers of viable oocysts in vitro. Laboratories must either use vertebrate animals to produce oocysts or rely on commercial sources which are extremely expensive. Even excluding the expense, oocysts must be purified from feces at which time they are often exposed to a variety of chemical insults including reagents such as potassium dichromate and ether. In addition, microbial contamination is normally eliminated before the parasites are utilized by the addition of 10% (v/v) commercial bleach, which surface sterilizes the oocysts. Thus, any method where we can obtain sufficient numbers of oocysts in vitro without the need for animal propagation or coliform contamination would open up a variety of avenues that are currently unavailable. These avenues include1) providing opportunities to a wider variety of laboratories to work on the parasite at decreased expense, 2) developing gene knock-outs and allelic replacements that could be propagated indefinitely, 3) performing metabolic labeling studies and looking at incorporation of label into the purifiable oocysts, and 4) more easily studying parasite development in vitro following exposure of oocysts to disinfectants without pre-exposing oocysts to solvents or reagents beforehand. This R21 proposal will attempt to solve the two problems outlined above: 1) find a commercially available, artificial, serum-free medium that permits good parasite growth in vitro; and 2) develop a system whereby we can routinely, easily, cleanly, and inexpensively generate sufficient numbers of oocysts in vitro without the need for experimental animals.